The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a number of biological responses to planar aromatic hydrocarbons (PAHs). Chemicals which interact with the AhR include a variety of environmental contaminants, such as dioxins, PCBs, PAHs and benzo(a)pyrene, as well as natural products, such as flavones and carbazoles.
One of the most potent agonists of the AhR is dioxin. The term dioxin, as commonly used by the news media, is shorthand for 2,3,7,8-tetrachlorodibenzo-p-dioxin (“TCDD”). TCDD is only one member (i.e. congener) of the polychlorinated dibenzo-p-dioxin family, of which there are 75 possible congeners whose structures vary according to the number and location of the chlorine atoms. A great number of isomers that differ in the chlorine substitution patterns exist for these three types of skeletal structures. Dioxins or dioxin-like compounds are environmental pollutants that have raised public concern because of their toxicity and their ability to remain in the environment for a long time. It has become clear that dioxins with their various toxic properties are contained in chemicals such as herbicides and insecticides, exhaust gases and fly ashes from garbage incineration facilities, waste water from papermills, etc. Dioxin induces a number of receptor-mediated toxic responses, including a severe wasting syndrome, epidermal hyperplasia and metaplasia, tumor promotion and thymic involution.
AhR gene was highly conserved in amphibian, fish, and mammalian during evolution. Specific environmental and endogenous compounds were found to bind on AhR and this complex subsequently binds to DRE sequence to activate AhR downstream gene expressions. The AhR resides primarily in the cytosol, where it is associated with its molecular chaperone, Hsp90. Upon binding to an agonist, the AhR dissociates from Hsp90, translocates to the nucleus and dimerizes with a structurally related protein, aryl hydrocarbon nuclear translocator (ARNT). This complex interacts with enhancer elements upstream of target promoters and up-regulates the transcription of a variety of xenobiotic metabolizing enzymes (e.g., the cytochrome P450 encoded by CYP1A1). The AhR and ARNT are both members of the basic helix-loop-helix-PAS superfamily. The helix-loop-helix domain serves as a dimerization surface for AhR and ARNT and also positions the basic alpha-helix within the major groove of B-DNA to enable specific interactions with target enhancer elements. The PAS domain functions as a dimerization surface, harbors a repressor region, and also contains regions required for binding agonist and forming interactions with Hsp90. The AhR is essential for the toxicity of dioxins and related chemicals. The AhR mediates the exquisite sensitivity of animals to dioxins, where as little as 2 ng/kg/day can yield striking adverse effects (Tracey D. Bradshaw and David R. Bell, Clinical Toxicology, 2009, 47, 632-642). After binding of dioxin to the AhR, the activated AhR translocates rapidly from the cytosol to the nucleus and forms a heterodimer with ARNT, causing cellular responses that lead to toxicity, carcinogenesis and teratogenesis (Barabra Oesch-Bartlomowicz et al. PNAS Jun. 28, 2005, vol. 102, no. 26, pp. 9218-9223). Neurotoxicity of endocrine disruptors has been recently suspected to be a cause of neurological disorders. The endocrine disruptors exhibit various toxicity such as reproduction toxicity, immunotoxicity and neurotoxicity, and a part of toxicity is known to be expressed through AhR mediated pathway. The aryl hydrocarbon receptor is conjectured to be related to neurotoxicity since expression of the aryl hydrocarbon receptor has been detected in brain tissue (Petersen, S. L. et al., J. Comp. Neurol. (2000), 427(3): 428-439). Correlation of expression of neurological functions and developmental formation with gene expression by the aryl hydrocarbon receptor has been also suggested. Chun-Hua Lin et al. suggest that neuronal activity may facilitate AhR-mediated calcium signaling, which in turn enhances AhR-mediated gene regulation and mediated maturation-dependent dioxin neurotoxicity (Journal of Neurochemistry, 2008, 104, pp. 1415-1429).
Environmental contaminants such as dioxin are detected not only in environmental samples from the air, soils, waters and sediments of rivers, harbors and ports around big cities, etc. but also in biological samples such as foods, blood, urine and mothers' milk. Since such a widespread contamination of the environment has been a big social problem, there is a pressing need to detect dioxin exposure in the environment. Dioxin-like compounds often occur as poorly defined mixtures of these compounds in a larger matrix of other materials that make their analysis and quantitation difficult. A number of reports disclose bioassays for detection environmental contaminants such as dioxin-like compounds. Postlind et al. describe a bioassay made by binding the 5′-flanking region of human CYP1A1 into a luciferase vector, and the resultant is transfected into human hepatoma cells (Response of Human CYP1-Luciferase Plasmids to 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Polycyclic Aromatic Hydrocarbons, Vol. 118, Toxicology and Applied Pharmacology, pp. 255-268 (1993)). U.S. Pat. No. 5,854,010 provides a recombinant cell line, the mouse H1L1.1 cell line, that is made by using genetic engineering techniques for inserting dioxin responsive elements upstream of a luciferase reporter gene, and then, transfecting the resultant recombinant expression plasmid, identified as pGudLuc1.1, into mouse hepatoma cells. The mouse H1L1.1 cell line is useful in a method for the quantitative analysis of polyaromatic hydrocarbons such as dioxin in samples.
The above-mentioned references use hepatoma cells as platform to detect dioxin. However, they cannot be used to evaluate the damage of dioxin to neuron cells. Therefore, the neuronal cell platform is more appropriate to screen ligands activating AhR pathway and to evaluate its impacts on neuronal tissues than that constructed in hepatocytes. There is a need to develop a neuronal cell platform in the detection of environmental pollutants and selection of drugs of treating neurological disorders.